Abstract
Darwin's finches are an iconic example of adaptive radiation and evolution under natural selection. Comparative genetic studies using embryos of Darwin's finches have shed light on the possible evolutionary processes underlying the speciation of this clade. Molecular identification of the sex of embryonic samples is important for such studies, where this information often cannot be inferred otherwise. We tested a fast and simple chicken embryo protocol to extract DNA from Darwin's finch embryos. In addition, we applied minor modifications to two of the previously reported PCR primer sets for CHD1, a gene used for sexing adult passerine birds. The sex of all 29 tested embryos of six species of Darwin's finches was determined successfully by PCR, using both primer sets. Next to embryos, hatchlings and fledglings are also impossible to distinguish visually. This extends to juveniles of sexually dimorphic species which are yet to moult in adult-like plumage and beak colouration. Furthermore, four species of Darwin's finches are monomorphic, males and females looking alike. Therefore, sex assessment in the field can be a source of error, especially with respect to juveniles and mature monomorphic birds outside of the mating season. We caught 567 juveniles and adults belonging to six species of Darwin's finches and only 44% had unambiguous sex-specific morphology. We sexed 363 birds by PCR: individuals sexed based on marginal sex specific morphological traits; and birds which were impossible to classify in the field. PCR revealed that for birds with marginal sex specific traits, sexing in the field produced a 13% error rate. This demonstrates that PCR based sexing can improve field studies on Darwin's finches, especially when individuals with unclear sex-related morphology are involved. The protocols used here provide an easy and reliable way to sex Darwin's finches throughout ontogeny, from embryos to adults.
Highlights
Accurate and rapid sex identification is an important step in many research projects
We aimed to amplify a section of the chromodomain helicase DNA binding 1 (CHD1) gene to identify the sex of nine species of Darwin’s finches
The mCHD1F/mCHD1R polymerase chain reaction (PCR) products were analysed on 2% agarose gel at 4V/cm for 90 minutes for optimal band separation (Fig 1A), but shorter running times (e.g., 60 min) were successful
Summary
Accurate and rapid sex identification is an important step in many research projects. Distinguishing sex by morphological traits like colouration and ornamentation can be straightforward in mature sexually dimorphic birds. Often such traits develop gradually and younger birds of both sexes look alike [1]. This makes visual sexing of nestlings, fledglings and young adults difficult to impossible. Visual sexing can be difficult or impossible in monomorphic species of birds In these cases, behavioural observations including singing, or morphometric measurements could be used, but these are not always applicable and can be expensive, inaccurate and time-consuming [4]. Cloacal protuberances in males are visible only during breeding and are not always clearly distinguishable [6]
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